Method for preventing or treating a lung disease caused by active oxygen and free radical injury

ABSTRACT

The present invention provides a preventive or therapeutic agent for tissue injury caused by active oxygens and free radicals, having for its effective component γ-L-glutamyl-L-cysteine ester derivative indicated in the following general formula: ##STR1## wherein, R is a straight chain, branched or cyclical hydrocarbon group having 1-10 carbon atoms, or a straight chain or branched hydrocarbon group having 1-carbons substituted with an aromatic group, or its oxidized dimer. 
     This compound has the effect of elevating glutathione levels in tissue and biochemically substitute for glutathione thereby performing preventive or therapeutic effects against ischemia-reperfusion tissue injury in the heart, liver and other organs, arrhythmia, as well as lung injury caused by active oxygens and free radicals.

This is a divisional of application Ser. No. 08/780,367 filed on Jan. 9,1997, now U.S. Pat. No. 5,750,507 which is a divisional of applicationSer. No. 08/487,634, filed Jun. 7, 1995, now U.S. Pat. No. 5,631,234,which is a continuation of application Ser. No. 08/225,914, filed Apr.11, 1994, now abandoned, which is a continuation of application Ser. No.08/955,713, filed Dec. 15, 1992, now abandoned.

TECHNICAL FIELD

The present invention relates to a preventive and therapeutic agent forischemia-reperfusion tissue injury, a preventive and therapeutic agentfor arrhythmia, and a preventive and therapeutic agent for various lungdiseases caused by impairment due to active oxygens and free radicals,having for its effective component a γ-L-glutamyl-L-cysteine esterderivative or an oxidized dimer obtained by dehydrogenation of twoidentical molecules of such.

BACKGROUND ART

Glutathione is present in almost all biological tissues. Glutathione, amajor intracellular reducing substance, plays important roles incatalysis, metabolism, transport and in cellular protection. Withrespect to cellular protection in particular, glutathione displays itsaction by (1) capturing and detoxifying harmful free radicals andperoxides produced in the body via the SH group of its own molecule, (2)reducing and deactivating active oxygens such as peroxides by means ofglutathione peroxidase, and (3) reacting with toxic compounds by meansof glutathione S-transferase and excreting those toxic compounds outsidethe cell in the form of glutathione conjugates, thereby playing roles inantioxidation, detoxification, protection against radiation injury andincreasing temperature resistance, etc.

Thus, when the level of tissue glutathione decreases due to variousdiseases, ageing and so on, the tissue becomes susceptible to injuries.In such cases, it is important to correct the levels of tissueglutathione to normal values in order to restore cell function. Inaddition, even in cases when tissue glutathione levels are normal, it isthought that increasing tissue glutathione levels can enhance cellprotective functions. In actuality, there are reports which state thatglutathione and several thiol compounds were effective when used for thepurpose of protecting cells from mutagenic substances and carcinogenicsubstances as well as reducing the size of animal liver tumors caused bysuch malignant substances.

Moreover, there has been growing attention in recent years focusing onthe correlation between depressed levels of glutathione and theoverproduction of active oxygens in relation to ischemia and infarctiondisorders of various tissues including the heart and brain, as well asvarious types of lung disorders.

In addition, it is gradually becoming clear that when ischemia iscanceled by restoration of blood flow, the formation of active oxygensis accelerated, which brings about even more remarkable decrease inglutathione, thereby resulting in the occurrence of more seriousdisorders. Similar disorders are observed during cessation orrestoration of blood flow during transplantation of various organs suchas heart, liver, lungs, pancreas and blood vessels. Those disorders alsobecome problem during incision and removal of organs. Active oxygens andreactive free radicals that are suspected to be the cause of disease aredetected both in the cytoplasm of cells that compose tissue and incellular organelles, especially in mitochondria that produce ATP, whichserves as the primary source of energy for cells. It is observed that,in mitochondria, the respiratory chain is the main source of generationof said reactive molecules and the concentration of such has becomeremarkably elevated during ischemia and reperfusion. In addition,extracellular active oxygens, that are produced on the cell membranesurface of leukocytes that gather at the lesion, are also considered tobe harmful to adjacent cells.

The important function of glutathione in the elimination of activeoxygens that form during ischemia-reperfusion was first reported in thestudies using animals for the disease model (for example, J. Neurochem.,Vol. 34, pp. 477-486, 1980; or, Curr. Surgery, January-February edition,pp. 31-33, 1988). Moreover, this was also recognized in human diseases(Circulation, Vol. 81, pp. 201-211, 1990). In each of these cases, thedegree of severity of the disease was considered to correlate with thedegree of consumption of glutathione.

In addition, it is also reported that in various lung diseases, theseverity of the disease state correlates with the decrease inglutathione on the surface of pulmonary epithelial cells (for example,Lung, Vol. 169, pp. 123-138, 1991).

Thus, the significance of replenishing glutathione from outside the bodyin the prevention and treatment of such diseases would be appreciated,while glutathione itself, the half-life of which in the blood whenadministered in short, i.e., several minutes, is not very effective inraising tissue glutathione levels. The reason for this is consideredthat glutathione itself is not efficiently taken up by cells, andglutathione must first be degraded and taken up by cells in the form ofpeptides and constituent amino acids followed by re-synthesis in thecell.

In an effort to overcome the above-mentioned problems, compounds havebeen discovered that are superior to glutathion with respect to theability to elevate tissue glutathione levels. Examples of such compoundsinclude 2-oxothiazolidine-4-carboxylate, γ-L-glutamyl-L-cysteine, andγ-L-glutamyl-L-cysteinyl-glycine ethyl ester (glutathione monoethylester). These compounds were discovered through experimentation usinghuman lymphoma cells or animals (for example, Curr. Top. Cell. Regul.,Vol. 26, pp. 383-394, 1985; or, Fed. Proc., Vol. 43, pp. 3031-3042,1984).

The compound represented with general formula (I): ##STR2## wherein R isan ethyl group, is known according to the description of JapaneseExamined Patent Publication No. 63-61302; and the compound wherein R isa straight chain, branched or cyclical hydrocarbon group having 3 to 10carbon atoms, or a straight chain or branched hydrocarbon group having 1to 5 carbon atoms substituted with an aromatic group is described inJapanese Unexamined Patent Publications No. 64-19059 and No. 2-258727;and the compound wherein R is a methyl group is known according to thedescription by Flohe, et al. in Z. Klin. Chem. u. Klin. Biochem., Vol.8, pp. 149-155 (1970), and is also described in Japanese UnexaminedPatent Publication No. 64-26516.

Although the documentation of Flohe, et al. indicates that theγ-L-glutamyl-L-cysteine derivative represented with general formula (I)wherein R is a methyl group serves as a substrate of glutathioneperoxidase and is used at 20-30% efficiency compared with glutathione,there are no other descriptions whatsoever that suggest other uses ofthis compound. In contrast, the previously listed Examined Publicationor three Unexamined Publications describe that theγ-L-glutamyl-L-cysteine ester derivative indicated in theabove-mentioned general formula (I), or its oxidized dimer, is an esterderivative of a glutathione biosynthesis intermediate and has favorablemembrane permeability; and that by means of being efficientlyincorporated into tissue and subsequently subjected to deesterificationfollowed by promptly biosynthesetic conversion into glutathione, thiscompound has the effect of elevating the level of tissue glutathione.These publications also describe that this compound is useful as atherapeutic agent for the treatment of liver disease, cataracts andkidney disease.

In addition, with respect to the possibility that, dependent on animals,tissues and dosages used in experiments, excess γ-L-glutamyl-L-cysteineester derivative incorporated may be present in tissue as unchanged SHcompound, the inventors of the present invention also reported thatγ-L-glutamyl-L-cysteine ester derivative has various desirableproperties: it serves as a good substrate for glutathione peroxidase,glutathione S-transferase and glutathione reductase in its originalform, while, unlike glutathione, it is resistant to breakdown byγ-glutamyl transpeptidase (SEIKAQ, Vol. 61, No. 9 (edition containingthe abstract of the 62nd meeting of the Japanese Biochemical Society),p. 800, title no. 1a-Ab08, 1989).

DISCLOSURE OF THE INVENTION

The inventors of the present invention discovered as to theγ-L-glutamyl-L-cysteine ester derivative represented with theabove-mentioned general formula (I), and its oxidized dimer that isobtained by dehydrogenation between the two of its identical molecule,which have the ability to elevate tissue glutathione levels as well asbiochemically substitute for glutathione, that, in coronaryischemia-reperfusion model using mongrel dogs, these derivatives (1)significantly suppress reduction of glutathione levels in mitochondriaof myocardium, (2) significantly suppress reduction of mitochondrialfunction in myocardium, and (3) significantly suppress the occurrence ofarrhythmia during reperfusion resulting in a significant decrease in themortality rate; and that, in liver ischemia-reperfusion model using ratsas well, these derivatives (1) suppress reduction of mitochondrialfunction during ischemia and promote restoration of such duringreperfusion, (2) significantly suppress reduction of glutathione levelsin liver tissue, (3) rapidly restore, during reperfusion, liver tissueATP that had been reduced during ischemia, (4) significantly suppresselevation of lipid peroxide levels in liver tissue followingreperfusion, and (5) significantly suppress elevation of theconcentration of adenine nucleotides in the hepatic vein immediatelyafter starting reperfusion owing to leakage from liver tissue, as wellas elevation of hepatocellular enzyme activity in the blood followingreperfusion. The inventors of the present invention also discovered inasthma model by inhalation of ovalbumin using guinea pigs that thesederivatives (1) prevent exacerbation of respiratory tract reactivity(decrease in log[Ach PC₂₀₀ ]), (2) prevent reduction of the number ofβ-receptors on the pulmonary membrane and decrease in adenylate cyclaseactivity, and (3) suppress elevation of xanthine oxidase activity in thelung tissue and bronchoalveolar lavage fluid.

Thus, based on the above-mentioned findings, the present inventionrelates to the application of γ-L-glutamyl-L-cysteine ester derivativeindicated in the following general formula (I): ##STR3## wherein R is astraight chain, branched or cyclic hydrocarbon group having 1-10 carbonatoms, or a straight chain or branched hydrocarbon group having 1-5carbons substituted with an aromatic group, or its oxidized dimerobtained by dehydrogenation between the two of its identical molecule,as an effective ingredient, to the prevention and treatment of tissueinjury occurring during ischemia-reperfusion of various tissuesincluding heart and liver: more concretely, diseases and tissue injurycaused by reduction, cessation or restoration of blood flow: forexample, various ischemic diseases such as angina pectoris, myocardialinfarction and cerebral infarction; post-operative injury followingincision, removal and transplantation of various organs; injuryoccurring during application of coronary bypass surgery, percutaneoustransluminal coronary angioplasty and anticoagulants; and arrhythmia; aswell as to the prevention and treatment of various lung diseasesincluding asthma caused by impairment due to active oxygens and freeradicals.

BRIEF DESCRITION OF THE DRAWINGS

FIGS. 1A and 1B are graphs respectively indicating the preventive andtherapeutic effects γ-L-glutamyl-L-cysteine ethyl ester of (γ-GCE) onthe cardiac muscle mitochondrial dysfunction, or in other words, thedecreased rate of state III oxygen consumption (St. III O₂), and thedecreased respiratory control index (RCI) in coronaryischemia-reperfusion model using dogs.

FIGS. 2A and 2B are graphs respectively indicating the preventive andtherapeutic effects of γ-GCE on the reduced mitochondrial functionduring ischemia and the recovery of such during reperfusion in liverischemia-reperfusion model using rats.

FIG. 3 is a graph indicating the preventive and therapeutic effects ofγ-GCE on the increase in airway responsiveness (Ach sensitivity) inovalbumin inhalation model using guinea pigs.

FIG. 4 is a graph indicating the preventive and therapeutic effects ofγ-GCE on the reduction in the number of β-receptors on pulmonarymembrane in ovalbumin inhalation model using guinea pigs.

FIG. 5 is a graph indicating the preventive and therapeutic effects ofγ-GCE on the decreased adenylate cyclase activity of the pulmonarymembrane in ovalbumin inhalation model using guinea pigs.

MODE OF OPERATION

In the above-mentioned general formula (I), R represents a straightchain, branched or cyclical hydrocarbon group having 1-10 carbon atoms,or a straight chain or branched hydrocarbon group having 1-5 carbonatoms substituted with an aromatic group. In addition, the oxidizeddimer refers to a dimer in which disulfide bonds (--S--S--) are formedfollowing dehydrogenation of two identical molecules of theabove-mentioned general formula (I).

The γ-L-glutamyl-L-cysteine ester derivative indicated in theabove-mentioned general formula (I), or its oxidized dimer, can bemanufactured according to the methods described in, for example,Japanese Examined Patent Publication No. 63-61302, Japanese UnexaminedPatent Publication No. 64-19059 and Japanese Unexamined PatentPublication No. 64-26516.

In the above-mentioned general formula (I), specific examples of Rinclude a methyl group, ethyl group, n-hexyl group, n-octyl group,isopropyl group, 2-methyl-2-propenyl group, cyclohexyl group and benzylgroup. Although the compound indicated in the above-mentioned generalformula (I) includes all γ-L-glutamyl-L-cysteine ester derivatives inwhich a particular R groups is bonded, typical examples of this compoundinclude γ-L-glutamyl-L-cysteine ethyl ester.

In the case of using the γ-L-glutamyl-L-cysteine ester derivative of theabove-mentioned general formula (I) or its oxidized dimer as a medicaldrug pertaining to the present invention, these compounds may be used intheir free form or in the form of pharmacologically acceptable acidic orbasic addition salts. When used in the form of a salt, the added acidicor basic groups may either be inorganic or organic compounds, and arenot limited in any manner as long as they are sufficiently effectivewhen used as a salt, and have no or low toxicity.

The compound of the above-mentioned general formula (I) or its oxidizeddimer may be administered orally, non-orally or via the respiratorytract in the desired form by mixing with pharmacologically acceptablecarriers, vehicles, solvents, diluents, coloring agents, preservatives,neutralizers and stabilizers for prevention and treatment pertaining tothe present invention.

Oral preparations can be either solid preparations such as tablets,granules, powders and capsules, or liquid preparations such as syrups,elixirs, emulsions and suspensions. In addition, non-oral preparationscan be in the form of injection preparations, suppositories or externalskin preparations. These preparations are made according to routinemethods by adding pharmacologically acceptable adjuvants to the compoundof the above-mentioned general formula (I) or its oxidized dimer.Moreover, these preparations can also be made into the form oftime-released preparations according to known methods.

Solid preparations for oral administration are made into powders bymixing the compound of the above-mentioned general formula (I) or itsoxidized dimer with a vehicle such as lactose, starch, cellulosecrystal, methyl cellulose, glycerine, sodium alginate, gum arabic,calcium hydrogenphosphate, meta-magnesium aluminum silicate, calciumlactate, sodium chloride, calcium carbonate and kaolin, or, ifnecessary, made into granules by adding a disintegrating agent such ashydroxypropyl cellulose, polyvinyl pyrrolidone, saccharose, sodiumalginate and sodium bicarbonate followed by granulation. Tablets aremade by forming these powders and granules into tablets as is, or byadding a glossing agent such as talc or magnesium stearate. Moreover,the above-mentioned granules or tablets can be coated with a base suchas methyl methacrylate copolymer or hydroxypropyl methyl cellulosephthalate to form an enteric coated preparation, or coated with ethylcellulose or a hardened oil to form a time-released preparation.Capsules can be formed into hard capsules by filling with powder orgranules, or formed into soft capsules by covering with a gelatin filmafter suspending or dissolving the compound of the above-mentionedgeneral formula (I) or its oxidized dimer in glycerine, polyethyleneglycol or olive oil and so on.

Liquid preparations for oral administration can be formed into a syrupby dissolving the compound of the above-mentioned general formula (I) orits oxidized dimer in water with a sweetener such as glycerine orsorbitol, an elixir by adding ethanol or essence, or an emulsion orsuspension by adding polysorbate 80, sodium carboxymethyl cellulose orgum arabic and so on.

Injection preparations are formed into single-dose or long or short-termcontinuous injection preparations for subcutaneous, intramuscular,intravenous or intraarterial injection by dissolving the compound of theabove-mentioned general formula (I) or its oxidized dimer in distilledwater for injection together with a pH regulator such as disodiumhydrogenphosphate, sodium dihydrogenphosphate, sodium hydroxide,hydrochloric acid, lactic acid or sodium lactate, an isotonic agent suchas glucose or sodium chloride, and an SH group stabilizer such as sodiumbisulfite, ascorbic acid or sodium ethylene diamine tetraacetate, andfilling into ampules or polyethylene or glass containers followingaseptic filtration. In addition, injection preparations of the type thatare prepared as needed can be made by freeze-drying in a vacuumfollowing the addition of dextrin, cyclodextrin, mannitol, gelatin andso on. In addition, the compound of the above-mentioned general formula(I) or its oxidized dimer may also be formed into an injectionpreparation contained in liposomes or microspheres in accordance withknown methods.

Suppositories can be made by heating and melting the compound of theabove-mentioned general formula (I) or its oxidized dimer withpolyethylene glycol, lanolin, mono-, di- or triglycerides of fatty acidsor cocoa butter, and coating with gelatin, etc., after either cooling toplasticize, or suspending or dissolving in soy bean oil, polyethyleneglycol and so on.

External skin preparations can be made by adding the compound of theabove-mentioned general formula (I) or its oxidized dimer topolyethylene glycol, white vaseline or liquid paraffin, etc., and may bein the form of an ointment, cream or gel and so on.

Preparations administered via the respiratory tract are administered inthe form of fine granules of the compound of the above-mentioned generalformula (I) or its oxidized dimer using routine inhalation methods. Itis desirable that the fine particles containing the drug as itseffective component be in the form of an aerosol or powder, and have aparticle size of 0.5-50 μm. Examples of devices that can be used toproduce the aerosol include ultrasonic and jet spray type nebulizers,and sprayers using lower alkanes or fluorinated alkanes as thepropellant. In addition, powders are administered using a simple inhalercoupled with spontaneous or forced inhalation.

Although there are no particular limitations on the concentration of thecompound represented with general formula (I) or its oxidized dimer inthe present medical drug preparation, in general, 0.1-70% by weight, andpreferably 1-50% by weight, is suitable for the concentration in thepreparation. In addition, although there are also no limitations on itsdosage, 0.01-5 g/day/patient, and preferably 0.1-2.5 g/day/patient, issuitable. With the exception of continuous injection, the number ofadministrations is normally 1-4 times per day.

EXAMPLES

Although the following provides a detailed explanation of the presentinvention using the Examples, the present invention is not limited bysaid Examples.

Example 1

Preventive and therapeutic effects of γ-L-glutamyl-L-cysteine ethylester (γ-GCE) on ischemia-reperfusion injury and accompanying arrhythmiain a coronary ischemia-reperfusion model using dogs.

Experimental Method

After anesthetizing 52 adult mongrel dogs of either sex weighing ca. 10kg by intraperitoneal administration of 50 mg/kg of sodiumpentobarbital, breathing tubes were immediately intubated endotracheallyand artificial ventilation was continued using a Harvard ventilator.Electrocardiogram electrodes were attached, and monitoring and recordingwere continued throughout the experiment. Following insertion ofcannulas in the right femoral vein and right femoral artery, thecannulas were used for continuous intravenous drip of physiologicalsaline to replenish water or administration of the test preparation, andmonitoring of arterial blood pressure, respectively. After performingleft thoracotomy in the fourth or fifth intercostal space, thepericardium was torn to expose the heart. Ischemia was induced byligating the left anterior descending coronary artery immediately distalto the first diagonal branch, while reperfusion was performed bysubsequently removing that ligation.

The animals were divided into four groups. Group 1 (n=9) was subjectedto ischemia for 2 hours, group 2 (n=17) was subjected to ischemia for 2hours followed by reperfusion for 1 hour, group 3 (n=10) was subjectedto ischemia for 2 hours followed by reperfusion for 1 hour plusintravenous injection of 10 mg/kg of γ-GCE just before reperfusion, andgroup 4 (n=16) was subjected to 2 hours of ischemia followed by 1 hourof reperfusion plus intravenous injection of 10 mg/kg of reducedglutathione (GSH, control drug) just before reperfusion.

Evaluation of ischemia-reperfusion injury was performed, followingcompletion of ischemia-reperfusion, by measuring (1) glutathione contentof mitochondria, and (2) mitochondrial function, and so on, at theischemic or reperfused area and the normal area of the heart.Mitochondria was obtained by removing the heart immediately after theend of ischemia or reperfusion, washing with cold physiological saline,promptly cutting out the ischemic or reperfused area and the normalarea, and immediately performing preparation from the respective areaaccording to the method described by Hatefi, et al. in Arch. Biochem.Biophys., Vol. 94, pp. 148-155, 1961.

The glutathione content of the mitochondria was measured by applying themethod described by Tietze in Anal. Biochem., Vol. 27, pp. 502-522, 1969to the supernatant obtained by immediately adding 1/10 vol of 27.5%(w/v) sulfosalicylic acid dihydrate aqueous solution to the preparedmitochondria suspension followed by protein denaturation andcentrifugation. Evaluation of mitochondrial function was performedaccording to the method described by Sugiyama, et al. in Am. Heart J.,Vol. 100, pp. 829-837, 1980, by polarographically measuring respiratoryability using an oxygen electrode and determining the rate of oxygenconsumption is state III (St. III O₂) and the respiratory control index(RCI).

Evaluation of arrhythmia was performed by analyzing theelectrocardiogram to determine the presence of arrhythmia, or in otherwords, the number of ventricular premature beats, as well as the totalduration of ventricular tachycardia, which was defined as more thanthree successive ventricular premature beats.

Results 1. Mortality Rate

Table 1 indicates the mortality rates during 2 hours of ischemiafollowed by 1 hour of reperfusion. In contrast to the mortality rateduring reperfusion being 0 in group 3, which was administered with γ-GCEjust before reperfusion, in group 2, which was not administered with adrug, and group 4, which was administered with GSH, ca. 50% of theanimals died due to ventricular fibrillation during reperfusion. Theabove results indicate that γ-GCE has excellent preventive andtherapeutic effects against sudden death caused by reperfusion injury.

Analyses of ischemia-reperfusion injury as well as arrhythmia describedin the following sections 2 and 3 were performed on a total of 32animals comprised of the surviving 8 animals in each group. Data wasexpressed in terms of the mean ± standard deviation, and evaluation ofsignificance was performed according to Dunnett's test. Cases in which Pwas less than 0.05 were considered to be statistically significant.

2. Preventive and Therapeutic Effects on Ischemia-ReperfusionReperfusion Injury

Table 2 indicates the glutathione contents of cardiac musclemitochondria at normal area and ischemic or reperfused area. The contentof reduced glutathione (GSH) and total glutathione, i.e., reduced form(GSH) plus oxidized form (GSSG), at ischemic or reperfused area,decreased to ca. 80% of the normal area after 2 hours of ischemia as canbe seen in group 1, and further decreased to ca. 60% after subsequent 1hour of reperfusion as can be seen in group 2. In contrast, in group 3,in which 10 mg/kg of γ-GCE was administered just before reperfusion,glutathione levels remained high, i.e., in the order of 85-88%, evenafter reperfusion. Not only did administration of γ-GCE significantlysuppress further reduction of glutathione during the reperfusion, but italso displayed a tendency to recover the glutathione reduction that hadoccurred during the 2 hours of ischemia. On the other hand, in group 4,which was administered with GSH, no such effects were observed. Theabove results indicate that γ-GCE has superior properties in membranepermeability and has excellent preventive and therapeutic effects on thereduction of glutathione level of intracellular organelle mitochondriaduring ischemia-reperfusion.

FIGS. 1A and 1B indicate mitochondrial function of cardiac muscle, atboth normal area and ischemic or reperfused area, i.e., the rate ofoxygen consumption in state III (St. III O₂), and the respiratorycontrol index (RCI), respectively. In group 3, in which 10 mg/kg ofγ-GCE was administered just before reperfusion, not only was the furtherreduction in mitochondrial function during reperfusion completelysuppressed, but also was the reduced function that had resulted duringischemia before administration of γ-GCE significantly improved. Incontrast, in group 4, which was administered with GSH, no protectiveeffects were observed whatsoever. The above results indicate that γ-GCEhas superior effects on prevention and treatment of coronaryischemia-reperfusion injury.

3. Preventive and Therapeutic Effects on Arrhythmia

Table 3 indicates the number of arrhythmia (in other words, theventricular premature beats) and the total duration of ventriculartachycardia, in which more than three successive ventricular prematurebeats occurred, during ischemia-reperfusion. As can be seen in group 2,which was not administered with a drug, ventricular premature beatsoccurred almost exclusively during the 60 minutes of reperfusion ratherthan during the 120 minutes of ischemia. This remarkable occurrence ofarrhythmia during reperfusion was strikingly suppressed in group 3,which was administered with 10 mg/kg of γ-GCE just before reperfusion,while, in group 4, which was administered with GSH, said occurrence wasnot suppressed at all. On the total duration of ventricular tachycardia,likely on the number of ventricular premature beats, γ-GCE remarkablysuppressed its increase during reperfusion, while GSH did not show sucheffect whatsoever. The above results indicate that γ-GCE has superiorpreventive and therapeutic effects against the occurrence of arrhythmiaaccompanying coronary ischemia-reperfusion injury.

                  TABLE 1                                                         ______________________________________                                        Mortality Rate of Dogs During Coronary Ischemia-                              Reperfusion Experiment (Number of dogs that died /                            Number of dogs in experiment)                                                             2 Hours of                                                                           1 Hour of                                                              Ischemia                                                                             Reperfusion                                                ______________________________________                                        Group 1       1/9                                                             Group 2       1/17     8/16                                                   Group 3       2/10     0/8                                                    Group 4       1/16     7/15                                                   ______________________________________                                         Group 1: 2 hours of ischemia                                                  Group 2: 2 hours of ischemia/1 hour of reperfusion                            Group 3: 2 hours of ischemia / 1 hour of reperfusion + Administration of      GCE just before reperfusion                                                   Group 4: 2 hours of ischemia / 1 hour of reperfusion + Administration of      GSH just before reperfusion                                              

                  TABLE 2                                                         ______________________________________                                        Glutathione Contents of Mitochondria in Cardiac Muscle                        Following Coronary Ischemia-Reperfusion in Dogs                                                     Ischemic or Re-                                                   Normal Area perfused Area                                           ______________________________________                                        GSH + GSSG                                                                    Group    1      4.03 ± 0.57                                                                              3.26 ± 0.51*                                          2      4.12 ± 0.40                                                                              2.53 ± 0.66**†                                 3      4.06 ± 0.52                                                                              3.57 ± 0.46§                                     4      3.94 ± 0.33                                                                              2.44 ± 0.57**††                GSH                                                                           Group    1      3.59 ± 0.46                                                                              2.87 ± 0.40*                                          2      3.67 ± 0.45                                                                              2.16 ± 0.64**†                                 3      3.68 ± 0.54                                                                              3.12 ± 0.40§                                     4      3.67 ± 0.37                                                                              2.17 ± 0.52**†                        GSSG                                                                          Group    1      0.44 ± 0.28                                                                              0.40 ± 0.17                                           2      0.45 ± 0.14                                                                              0.37 ± 0.15                                           3      0.38 ± 0.09                                                                              0.45 ± 0.15                                           4      0.27 ± 0.08                                                                              0.27 ± 0.09                                  ______________________________________                                         Values are expressed as mean ± standard deviation                          *P < 0.05; **P < 0.01 versus normal area of corresponding group               tP < 0.05; ttP < 0.01 versus ischemic area of Group 1                         SSP < 0.01 versus reperfused area of Group 2                             

                  TABLE 3                                                         ______________________________________                                        Arrhythmia during Coronary Ischemia and                                       Reperfusion in Dogs                                                                               Total Duration of                                         Number of Ventricular                                                                             Ventricular                                               Premature Beats     Tachycardia (sec)                                                      1 Hour of            1 Hour of                                   2 Hours of   Reperfu-   2 Hours of                                                                              Reperfu-                                    Ischemia     sion       Ischemia  sion                                        ______________________________________                                        Group 1                                                                              30 ± 31           3 ± 6                                          Group 2                                                                              11 ± 15                                                                              1376 ± 956                                                                            0 ± 1                                                                              1029 ± 671                             Group 3                                                                              40 ± 51                                                                               32 ± 30*                                                                             1 ± 2                                                                               3 ± 5*                                Group 4                                                                              42 ± 43                                                                              1731 ± 1574                                                                           1 ± 2                                                                              983 ± 959                              ______________________________________                                         Values are expressed as the mean ± standard deviation.                     *P < 0.01 versus 1 hour of reperfusion of Group 2                        

Example 2

Preventive and therapeutic effects of γ-GCE on ischemia-reperfusioninjury in a liver ischemia-reperfusion model using rats.

Experimental Method

Male Wister rats weighing 250-300 g were fasted with free access towater 24 hours before the experiment. Seventy-two rats were divided intothree groups of 24 animals each, i.e., the control group, the GSHadministration group and the γ-GCE-administration group. Each rat wasanesthetized by intraperitoneal administration of 40 mg/0.8 ml/kg ofsodium pentobarbital, and the femoral vein was cannulated. In the GSHgroup and the γ-GCE group, 50 mg/kg of GSH or γ-GCE respectively wereadministered intravenously. Physiological saline was then infused forvolume replacement in each group, as a continuous intravenous infusionat a rate of 0.8 ml/hr with an injection pump throughout the experiment.Thirty minutes after starting injection of physiological saline, theabdomon was opened by a midline incision and the liver hilus wasexposed. According to the method described by Hasselgren, et al. in J.Surg. Res., Vol. 34, pp. 409-414, 1984, all vessels to the left andmedian lobes of the liver, more specifically, hepatic artery, portalvein and the bile duct, were clamped to produce an ischemic state for 2hours. Since blood vessels to the remaining parts of the liver were notoccluded by this method, poral stasis could be avoided which was ofspecial relevance for circulation stability in rats. Thereafter,occlusion of all vessels was released by removing the clamp to allowreperfusion for 1 hour. The abdominal walls were closed during ischemiaand reperfusion.

Using 18 rats in each group, the livers of 6 animals from each groupwere removed before ischemia, after 2 hours of ischemia, and after 1hour of reperfusion, respectively. A portion of the liver from eachanimal was rapidly frozen in liquid nitrogen and used for measurement ofglutathione, adenine nucleotides and lipid peroxide. From the remainingportion of the liver, mitochondria was immediately extracted accordingto the method described by Hogeboom and Schneider in the J. Biol. Chem.,Vol. 172, p. 619, 1948. In other experiments, immediately after startingreperfusion, blood from the hepatic vein was collected using 6 rats fromeach group. These blood samples were then used to measure leaked adeninenucleotides. In addition, after 1 hour of said reperfusion, bloodsamples were also drawn, which were then used to measure the activity ofthe leaked liver cell enzymes.

Evaluation of ischemia-reperfusion injury was performed by measuringliver tissue (1) mitochondrial function, (2) glutathione concentration,(3) adenine nucleotides concentration, and (4) lipid peroxideconcentration, as well as liver tissue derived (5) adenine nucleotidesconcentration in the hepatic vein immediately after startingreperfusion, and (6) liver cell enzyme activities in the blood after 1hour of reperfusion. More specifically, the mitochondrial function in(1) was measured according to the method described by Chance, et al. inAdv. Enzymol., Vol. 17, p. 65, 1956. In addition, the quantitativedetermination of glutathione in (2) was performed using the same methodas Experiment 1 on supernatant obtained by adding 10 vol/g of tissueweight of 2.5% (w/v) sulfosalicylic acid dihydrate aqueous solution tothe liver tissue followed by homogenization and centrifugation. Inaddition, the quantitative determination of adenine nucleotides in (3)was performed by neutralizing the perchloric acid extract of the livertissue with potassium hydroxide followed by removal of the precipitateand applying to an ion exchange HPLC column according to the methoddescribed by Sugino, et al. in J. Chromatogr., Vol. 361, pp. 427-431,1986. In addition, the measurement of lipid peroxide in (4) wasperformed according to the thiobarbiturate method described by Ohkawa,et al. in Anal. Biochem., Vol. 95, p. 351, 1979. In addition, thequantitative determination of adenine nucleotides in (5) was performedby centrifuging blood from the hepatic vein to obtain the serum andremoving the protein in the serum by centrifugation using a microfilter,followed by applying this to an HPLC column in the same manner as (3).In addition, the measurement of the activities of AST, ALT and LDH, etc.in (6) was performed using Uni-Kit (Chugai Co., Ltd., Tokyo, Japan).

All results were expressed as mean ± standard deviation. Evaluation ofsignificance was performed using Dunnett's test. Cases in which P wasless than 0.05 were considered to be statistically significant.

Results

FIGS. 2A and 2B indicate liver tissue mitochondrial function. γ-GCEsignificantly suppressed reduction of St. III O₂ caused by ischemia, andalso significantly promoted recovery of mitochondrial function duringreperfusion. In contrast, GSH did not show any protective effectswhatsoever.

Table 4 indicates changes in glutathione concentration in liver tissue.In the control and GSH administration groups, glutathione concentrationsignificantly decreased after reperfusion to ca. 60% and 70% of thelevel prior to ischemia, respectively. In contrast, in the γ-GCEadministration group, no significant decrease was observed throughoutthe experiment. Moreover, liver glutathione concentration in the γ-GCEgroup were significantly higher than in the control group or GSHadministration group throughout the experiment.

Table 5 indicates changes in adenine nucleotides concentration in livertissue. Two hours of ischemia brought about a remarkable decrease in ATPand ADP, while conversely, causing a remarkable increase in AMP.Although γ-GCE did not show any effects on the reduction of ATP causedby ischemia, it significantly promoted restoration of ATP followingreperfusion. GSH did not show any promotive effects on the restorationof ATP

                  TABLE 4                                                         ______________________________________                                        Changes in Glutathione Concentrations in Liver Tissue                         During Liver Ischemia and Reperfusion in Rats                                          Glutathione (μmol/g of liver wet weight)                                   GSH      GSSG        GSH + GSSG                                      ______________________________________                                        Before Ischemia                                                               Control group                                                                            4.92 ± 0.58                                                                           0.30 ± 0.05                                                                            5.22 ± 0.57                              GSH group  4.91 ± 0.35                                                                           0.38 ± 0.06                                                                            5.29 ± 0.38                              γ-GCE group                                                                        5.65 ± 0.30*                                                                          0.39 ± 0.07*                                                                           6.04 ± 0.35*                             After 2 Hours                                                                 Ischemia                                                                      Control group                                                                            4.25 ± 0.48                                                                           0.26 ± 0.04                                                                            4.51 ± 0.48                              GSH group  4.35 ± 0.65                                                                           0.35 ± 0.04*                                                                           4.40 ± 0.68                              γ-GCE group                                                                        5.48 ± 0.42**                                                                         0.39 ± 0.01**                                                                          5.87 ± 0.43**                            After 1 Hour                                                                  Reperfusion                                                                   Control group                                                                            3.01 ± 0.53##                                                                         0.20 ± 0.05                                                                            3.22 ± 0.57##                            GSH group  3.40 ± 0.60##                                                                         0.37 ± 0.05**                                                                          3.77 ± 0.64##                            γ-GCE group                                                                        4.97 ± 0.25**                                                                         0.54 ± 0.09**                                                                          5.50 ± 0.26**                            ______________________________________                                         All values are expressed as the mean of 6 analytical values ± standard     deviation.                                                                    *P < 0.05;                                                                    **P < 0.01 versus control group                                               ##P < 0.01 versus before ischemia of the respective group                

                  TABLE 5                                                         ______________________________________                                        Changes in Adenine Nucleotides Concentrations in Liver                        Tissue During Liver Ischemia and Reperfusion in Rats                                     Adenine Nucleotides (μmol/g wet liver weight)                                                     ATP + ADP +                                            ATP   ADP      AMP     AMP                                         ______________________________________                                        Before Ischemia                                                               Control group                                                                              2.71 ±                                                                             0.85 ±                                                                              0.19 ±                                                                           3.75 ±                                              0.15    0.07     0.02  0.18                                      GSH group    2.57 ±                                                                             0.65 ±                                                                              0.18 ±                                                                           3.40 ±                                              0.16    0.06     0.01  0.20*                                     γ-GCE group                                                                          2.69 ±                                                                             0.57 ±                                                                              0.13 ±                                                                           3.39 ±                                              0.15    0.05     0.01  0.16*                                     After 2 Hours Ischemia                                                        Control group                                                                              0.05 ±                                                                             0.14 ±                                                                              0.83 ±                                                                           1.02 ±                                              0.01    0.02     0.08  0.10                                      GSH group    0.04 ±                                                                             0.13 ±                                                                              0.73 ±                                                                           0.90 ±                                              0.01    0.02     0.11  0.11                                      γ-GCE group                                                                          0.06 ±                                                                             0.17 ±                                                                              0.87 ±                                                                           1.10 ±                                              0.01    0.03     0.11  0.10                                      After 1 Hour Reperfusion                                                      Control group                                                                              1.06 ±                                                                             0.27 ±                                                                              0.09 ±                                                                           1.42 ±                                              0.08    0.02     0.01  0.09                                      GSH group    1.13 ±                                                                             0.38 ±                                                                              0.15 ±                                                                           1.66 ±                                              0.13    0.04**   0.02  0.16                                      γ-GCE group                                                                          1.70 ±                                                                             0.38 ±                                                                              0.12 ±                                                                           2.21 ±                                              0.02**  0.03**   0.01  0.03**                                    ______________________________________                                         All values are expressed as the mean of 6 analytical values ± standard     deviation.                                                                    *P < 0.05;                                                                    **P < 0.01 versus control group                                          

Lipid Peroxide Concentrations

Concentration of liver lipid peroxide following reperfusion (9.7±0.7nmol/mg protein) significantly increased (P<0.01) in comparison toconcentration after 2 hours of ischemia (6.4±0.6 nmol/mg protein).Administration of γ-GCE significantly suppressed this increase (6.56±0.6nmol/mg protein, P<0.01). However, administration of GSH did not haveany protective effects whatsoever (9.1±2.0 nmol/mg protein).

Leakage of Adenine Nucleotides and Liver Cell Enzymes

As indicated in Table 6, γ-GCE significantly reduced the leakage ofadenine nucleotides into the hepatic vein observed immediately afterstarting reperfusion by over 50%, and in addition, as indicated in Table7, γ-GCE significantly reduced leakage of enzymes from liver cells aswell observed in the blood following 1 hour of reperfusion.Administration of GSH did not significantly mitigate the leakage ofeither adenine nucleotides or liver cell enzymes.

                  TABLE 6                                                         ______________________________________                                        Adenine Nucleotides Concentrations in Hepatic Vein                            Immediately After Starting Reperfusion                                                    Adenine Nucleotides                                                           (μmol/L serum)                                                 ______________________________________                                        Control group 4.23 ± 2.04                                                  GSH group     3.59 ± 1.28                                                  γ-GCE group                                                                            1.96 ± 0.80*                                                ______________________________________                                         All values are expressed as the mean of 6 analytical values ± standard     deviation.                                                                    *P < 0.05 versus control group                                           

                  TABLE 7                                                         ______________________________________                                        Serum AST, ALT and LDH Concentrations                                         After 1 Hour of Reperfusion                                                          IU/L                                                                          AST       ALT         LDH                                              ______________________________________                                        Control group                                                                          4805 ± 1260                                                                            2957 ± 936                                                                             39928 ± 14416                             GSH group                                                                              3914 ± 1102                                                                            2337 ± 571                                                                             26435 ± 12191                             γ-GCE group                                                                       2071 ± 625**                                                                            924 ± 330**                                                                           14678 ± 2763**                           ______________________________________                                         All values are expressed as the mean of 6 analytical values ± standard     deviation. Abbreviations; AST: Aspartate Amino Transferase, ALT: Alanine      Amino Transferase, LDH: Lactate Dehydrogenase.                                **P < 0.01 versus control group.                                         

The above results indicate that γ-GCE has superior preventive andtherapeutic effects against liver ischemia-reperfusion injury.

Example 3

Preventive and therapeutic effects of γ-GCE on the increase in airwayresponsiveness in an ovalbumin inhalation model using guinea pigs.

Experimental Method

Male Hartley guinea pigs weighing 350-400 g were divided into threegroups, i.e., the control group, the ovalbumin (OA) group and theOA+γ-GCE group. The control group was allowed to inhale an aerosol ofphysiological saline for 7-8 minutes per day for 10 consecutive days.The OA group was allowed to inhale an aerosol of 2% OA in the samemanner. The OA+γ-GCE group was allowed to similarly inhale an aerosol of2% OA together with intraperitoneal administration of 10 mg/kg of γ-GCEtwice daily for 10 days.

Evaluation of efficacy was performed by measuring (1) the minimumprovocative concentration of acetylcholine (Ach) at which therespiratory resistance reached more than 200% of baseline (AchProvocative Concentration 200) both before and after inhalation for 10days, together with measuring (2) the number of pulmonary membraneβ-receptors and adenylate cyclase activity, and (3) xanthine oxidaseactivities in lung tissue, bronchoalveolar lavage fluid and serum,following 10 days of inhalation.

Each of the measurements were made using 6 animals in each group, andthe results were expressed as mean ± standard deviation. Evaluation ofsignificance was performed using Dunnett's test. Cases of P being lessthan 0.05 were considered to be statistically significant.

Results

FIG. 3 indicates the changes in log[Ach PC₂₀₀ ] as the indicator ofairway responsiveness before and after inhalation. FIGS. 4 and 5indicate the number of pulmonary membrane β-receptors and pulmonarymembrane adenylate cyclase activity after 10 days of inhalation,respectively. Table 8 indicates xanthine oxidase activities in lungtissue, bronchoalveolar lavage fluid and serum following 10 days ofinhalation.

                  TABLE 8                                                         ______________________________________                                        Xanthine Oxidase Activities in Lung Tissue;                                   Bronchoalveolar Lavage Fluid and Serum                                                             Bronchoalveolar                                                   Lung Tissue Lavage       Serum                                       Test Group                                                                             (mU/g tissue)                                                                             Fluid (mU/ml)                                                                              (mU/ml)                                     ______________________________________                                        Control  7.35 ± 6.48                                                                            2.85 ± 1.17                                                                             3.51 ± 1.15                              OA       49.1 ± 11.7*                                                                           12.6 ± 3.16                                                                             11.5 ± 2.66*                             GCE+ γ                                                                           33.3 ± 4.02*#                                                                          9.43 ± 0.95*.sup.†                                                               10.0 ± 1.77*                             ______________________________________                                         All values are expressed as mean ± standard deviation.                     *P < 0.01 versus control group                                                #P < 0.01 versus OA group                                                     .sup.† P < 0.05 versus OA group                                   

Administration of γ-GCE inhibited (1) the remarkable increase in airwayresponsiveness (remarkable decrease in log[Ach PC₂₀₀ ]), and (2) theremarkable reduction in membrane β-receptors (38% reduction) as well asthe remarkable reduction in membrane adenylate cyclase activity (38%reduction during stimulation by isoproterenol, 28% reduction atbaseline), and, despite inhalation of OA, maintained values of suchequal to those of no OA inhalation. In addition, xathine oxidase valuesof the OA+γ-GCE group were also significantly lower than those of the OAgroup, in lung tissue and bronchoalveolar lavage fluid and so on.

The mechanism of the occurrence of airway hyperresponsiveness (asthma)due to OA inhalation was considered to involve the conversion ofxanthine dehydrogenase to xanthine oxidase by some mechanismaccompanying OA inhalation, followed by the production of oxygenradicals, which might in turn induce marked acceleration of enzymeconversion and exacerbation of tissue damage including deterioration ofreceptor function. The above results indicate that γ-GCE has superiorpreventive and therapeutic effects against various lung diseasesincluding asthma resulted from active oxygens and free radical injury,by inhibiting such vicious circle as a radical scavenger.

What is claimed is:
 1. A method for preventing or treating asthma causedby active oxygen and free radical injury, comprising administering to amammal in need thereof a lung disease preventing or treating effectiveamount of: a γ-L-glutamyl-L-cysteine ester compound of formula (I):##STR4## wherein R is a straight chain, branched or cyclic hydrocarbongroup having 1-10 carbon atoms, or a straight chain or branchedhydrocarbon group having 1-5 carbon atoms substituted with an aromaticgroup; or an oxidized dimer obtained by dehydrogenation between twoγ-L-glutamyl-L-cysteine esters having formula (I).
 2. The method ofclaim 1, wherein R is a lower alkyl.
 3. The method of claim 2, wherein Ris an ethyl group.
 4. The method of claim 1, wherein said effectiveamount of said compound or said dimer is between 0.01-5 g/day.
 5. Themethod of claim 4, wherein said effective amount of said compound orsaid dimer is between 0.01-2.5 g/day.